Listeria and Shigella hijack the actin cytoskeleton of their host cell and use it to spread from cell to cell. Act A, a Listeria surface protein is the only bacterial protein required for assembly of the actin motor and it interacts directly with VASP, a host cytoskeletal protein. This interaction requires at least one of the 4 tandem proline repeat motifs found in ActA. Profilin, an actin binding protein that has several potent effects on actin assembly, binds directly to VASP. This interaction is mediated by different tandem proline repeat motifs found on VASP, and can be blocked by these proline motifs in in vivo assays. Act A, profilin, and VASP accumulate at the interface between the bacterial surface and the growing actin tail. This proposal aims to identify mechanisms by which these protein:protein interactions enable Listeria and Shigella to assemble actin-based motors at their surfaces. The investigator proposes to investigate the nature of these interactions between ActA, VASP, and profilin through a series of genetic alterations of the VASP binding domain on ActA combined with cell biological and biochemical assays. this is a research area that will deepen understanding not only of particular pathogenic processes, but also of the molecular basis for regulation of the actin cytoskeleton to achieve cell motility. There are three specific aims. Aim 1 is to study the binding of ActA, the bacterial surface protein which is the only bacterial protein required for bacterial motility to a host cell protein VASP (vasodilator-stimulated phosphoprotein), already known to bind to ActA and also necessary for motility. Listeria must then, attract VASP to ActA. ActA has four oligoproline sequences separated by stretches of amino acids, (the first, DFPPPPTDE and the other 3 are similar, one has only 3 P interrupted with I); thus prolines with an aromatic group at the start and flanked by negatively charged residues. This is the critical VASP binding site and all 4 are thought to operate but this is not proven. Using PCR site-direct mutagenesis and bacterial expression, the PI proposes to study binding by gel filtration and analytical centrifugation to identify the primary structural determinants and binding constants for the complex between ActA and VASP. Oligoproline analogues will be prepared and studied as competitive inhibitors of complex formation. Aim 2 combines two aims: the binding of vinculin to VASP and ActA function in vivo. The thought is that Listeria must attract VASP from the vinculin binding in the host. Therefore in vitro binding studies will determine the relative affinity of VASP to vinculin vs. ActA. The rank order of peptide analogue inhibition of ActA-VASP and Vinculin-ActA binding will also be studied in vivo by microinjection and time-lapse video microscopy. Listeria ActA mutants will be prepared with a shuttle vector PMK4 and homologous recombinations made with defined changes in the oligoproline repeat sequences. The ability of each mutant bacterium to generate actin tails, move within and spread from cell to cell and to cause disease in mice will be studied. Aim 3 is to study the binding of VASP to profilin and the interaction of this complex with ActA. The idea is that profilin binding to VASP or VASP-ActA may alter the ability of profilin to enhance ATP/ADP exchange on actin monomers.